Metabolic, Cardiovascular and Inflammatory Disease Genomics Branch, National Human Genome Research Institute (NHGRI), Bethesda, MD, USA.
Department of Laboratory Medicine, Center for Genetic Medicine Research, Children's National, Washington, DC, USA.
J Clin Immunol. 2020 Aug;40(6):917-926. doi: 10.1007/s10875-020-00817-3. Epub 2020 Jul 8.
Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disorder that manifests with fever, early-onset vasculitis, strokes, and hematologic dysfunction. This study aimed to identify disease-causing variants by conventional Sanger and whole exome sequencing in two families suspected to have DADA2 and non-confirmatory genotypes. ADA2 enzymatic assay confirmed the clinical diagnosis of DADA2. Molecular diagnosis was important to accurately identify other family members at risk.
We used a variety of sequencing technologies, ADA2 enzymatic testing, and molecular methods including qRT-PCR and MLPA.
Exome sequencing identified heterozygosity for the known pathogenic variant ADA2: c.1358A>G, p.Tyr453Cys in a 14-year-old female with a history of ischemic strokes, livedo, and vasculitis. No second pathogenic variant could be identified. ADA2 enzymatic testing in combination with quantitative RT-PCR suggested a loss-of-function allele. Subsequent genome sequencing identified a canonical splice site variant, c.-47+2T>C, within the 5'UTR of ADA2. Two of her unaffected siblings were found to carry the same two pathogenic variants. A homozygous 800-bp duplication comprising exon 7 of ADA2 was identified in a 5-year-old female with features consistent with Diamond-Blackfan anemia (DBA). The duplication was missed by Sanger sequencing of ADA2, chromosomal microarray, and exome sequencing but was detected by MLPA in combination with long-read PCR sequencing. The exon 7 duplication was also identified in her non-symptomatic father and younger sister.
ADA2 pathogenic variants may not be detected by conventional sequencing and genetic testing and may require the incorporation of additional diagnostic methods. A definitive molecular diagnosis is crucial for all family members to make informed treatment decisions.
腺苷脱氨酶 2 缺乏症（DADA2）是一种常染色体隐性疾病，其特征为发热、早发性血管炎、中风和血液功能障碍。本研究旨在通过对两例疑似 DADA2 且基因型不确定的家系进行常规 Sanger 测序和全外显子测序，以鉴定致病变异。ADA2 酶活性测定证实了 DADA2 的临床诊断。分子诊断对于准确识别其他有风险的家族成员至关重要。
我们使用了多种测序技术、ADA2 酶活性测定以及包括 qRT-PCR 和 MLPA 在内的分子方法。
外显子组测序发现 14 岁女性存在已知致病性变异 ADA2：c.1358A>G，p.Tyr453Cys，该女性有缺血性中风、皮肤紫斑和血管炎病史。未能鉴定出第二种致病性变异。ADA2 酶活性测定结合定量 RT-PCR 提示存在功能丧失等位基因。随后的基因组测序在 ADA2 的 5'UTR 中发现了一个典型的剪接位点变异 c.-47+2T>C。她的两名无病兄弟姐妹均携带这两种致病性变异。一名 5 岁女性存在与 Diamond-Blackfan 贫血（DBA）一致的特征，携带一个 800bp 的 ADA2 外显子 7 纯合重复。该重复通过 ADA2 的 Sanger 测序、染色体微阵列和外显子组测序均未能检出，但通过 MLPA 结合长读 PCR 测序检测到。该外显子 7 重复也在她无症状的父亲和妹妹中检出。
ADA2 致病性变异可能无法通过常规测序和基因检测检出，可能需要纳入其他诊断方法。明确的分子诊断对于所有家庭成员做出知情的治疗决策至关重要。
